1. Field of the Invention
The field of invention relates to immunological reagents and methods for detecting the expression of specific antigens. Specifically, the invention relates to a monoclonal antibody which detects a variant of the membrane glycoprotein CD44. The immunological reagent of the present invention is useful as a diagnostic tool for detecting malignant transformation, assessing metastatic potential and for diagnosing inflammatory diseases.
2. Description of the Background Art
Cell-cell and cell-matrix interactions are of fundamental importance to multicellular organisms in controlling growth, differentiation and in the migration of cells. CD44 is one of the molecules known to be involved in these adhesion-dependent processes. In man, CD44 was independently discovered by several groups using antibodies against molecules referred to as "brain-granulocyte-T lymphocyte antigen"; "human medullary thymocyte antigen"; "Lutheran inhibitor related antigen"; "p85"; "phagocytic glycoprotein-1"; "Hermes-antigen"; "extracellular matrix receptor type III"; and "hyaluronate receptor" (Carter, W. G. et al., J. Biol. Chem. 263:4193-4201 (1988); Dalchau, R. et al., Eur. J. Immunol. 10:745-749 (1980); Haynes, B. F. et al., J. lmmunol. 131:1195-1200 (1983); Isacke, C. M. et al., Immunogenetics 23:326-332 (1986); Jalkanen, S. et al., Eur. J. Immunol. 16:1195-1202 (1986); Letarte M. et al., Mol. Immunol 22:113-124 (1985); Telen, M. J. et al., J. Clin. Invest. 71:878-1886 (1983); Underhill, C. B. et al., J. Biol. Chem. 262:1142-13146 (1987)). Subsequently, these antibodies were shown to identify the same molecule, CD44 (Culty, M. et al., J. Cell Biol. 111:2765-2774 (1990); Gallatin, W. M. et al., Proc. Natl. Acad. Sci. USA 86:4654-4658 (1989); Omary, M. B. et al., Immunogenetics 27:460-464 (1988); Picker, L. J. et al., J. Immunol. 142:2046-051 (1989)).
CD44 is a multifunctional glycoprotein involved in: lymphocyte-endothelial cell interactions; adhesion of cells to extracellular matrix proteins; lymphohematopoiesis; homotypic adhesion; T cell activation and adherence; cytokine release; metastasis and the lateral movement of cells (Haynes, B. F. et at., Cancer Cells 3:347-350 (1991); Haynes, B. F. et at., Immunol. Today 10:423-428 (1989); Jalkanen, S. T. et al., J. Cell. Biol. 105:983-990 (1987); Jalkanen, S. et al., Eur. J. Immunol. 16:1195-1202 (1986); Oppenheimer-Marks, N. et al., J. Immunol. 145:140-148 (1990); Picker, L. J. et al., J. Cell. Biol. 109:927-938 (1989); Aruffo, A. et al., Cell 61:1303-1313 (1990); Carter, W. G. et al., J. Biol. Chem. 263:4193-4201 (1988); Jalkanen, S. et al., J. Cell. Biol. 116:817-825 (1992); Miyake, K. et al., J. Exp. Med. 172:69-75 (1990); Underhill, C. B. et al., J. Biol. Chem. 262:1142-13146 (1987)); Miyake, K. et al., J. Exp. Med. 171:477-488 (1990); Belitsos, P. C. et al., J. Immunol. 144:1661-1670 (1990); St. John, T. et al., Cell 60:45-52 (1990); Arch, R. et al., Science 257:682-685 (1992); Denning, S. M. et al., J. Immunol. 144:7-15 (1990); Hale, L. P. et al., J. Immunol. 143:3944-3948 (1989); Huet, S. et al., J. Immunol. 143:798-801 (1989); Kalomiris, E. L. et al., J. Cell Biol. 106:319-327 (1988); Rothman, B. L. et al., J. Immunol. 147:2493-2499 (1991); Seth, A. et at., Proc. Natl. Acad. Sci. USA 88:7877-7881 (1991); Shimizu, Y. et al., J. Immunol. 143:2457-2463 (1989); Webb, D. S. A. et al., Science 249:1295-1297 (1990); Jacobson, K. et al., J. Cell Biol. 99:1613-1623 (1984)).
CD44 is widely distributed among several hematopoietic and nonhematopoietic cells including all subsets of leukocytes, erythrocytes, many types of epithelial cells, fibroblasts, smooth muscle cells and glial cells of the central nervous system (Carter, W. G. et al., J. Biol. Chem. 263:4193-4201 (1988); Dalchau, R. et al., Eur. J. Immunol. 10:745-749 (1980); Haynes, B. F. etal., J. Immunol. 131:1195-1200 (1983); Isacke, C. M. etal., Immunogenetics 23:326-332 (1986); Jalkanen, S. et al., Eur. J. Immunol. 16:1195-1202 (1986); Letarte M. et al., Mol. Immunol 22:113-124 (1985); Lucas, M. G. et al., Blood 735:596-600 (1989); Picker, L. J. et al., J. Cell. Biol. 109:927-938 (1989); Telen, M. J. et al., J. Clin. Invest. 71:878-1886 (1983)). Most hematopoietic cells, fibroblasts and glial cells predominantly express a 90 kD form of CD44. Lymphocytes also express a 180 kD form which represents a chondroitin sulfate modification of the 90 kD backbone (Jalkanen, S. et al., J. Immunol. 141: 16 15-1623 (1988)). In contrast, the CD44 antigen in epithelial cell lines is considerably larger (140-160 kD), and still larger forms, up to 230 kD have been described (Brown, T. A. et al., J. Cell Biol. 113:207-221 (1991); Omary, M. B. et al., Immunogenetics 27:460-464 (1988); Picker, L. J. et al., J. Cell. Biol. 109:927-938 (1989)).
Recently, the molecular basis underlying the biochemically distinct forms of CD44 has been resolved. Molecular cloning of human CD44 from lymphoid lines revealed a gene which encodes an integral membrane glycoprotein having an N-terminal extracellular region, a short hydrophobic transmembrane region and a cytoplasmic tail (Goldstein, A. L. et al., Cell 56:1063-1072 (1989); Stamenkovic, I. et al., Cell 56:1057-1062 (1989)). Subsequently, the structure of an epithelial form of 150 kD from keratinocytes and carcinoma cell lines was analyzed (Brown, T. A. et al., J. Cell Biol. 113:207-221 (1991); Stamenkovic, I. et al., EMBO J. 10:343-348 (1991)). The epithelial form was found to contain an additional stretch of 132 amino acids inserted in the membrane proximal part of the peptide backbone common to both the lymphocyte and epithelial forms. Forms containing the same 132 amino acid sequence or a shorter part of it were also found in hematopoietic cells (Dougherty, G. J. et al., J. Exp. Med. 174:1-5 (1991)).
In both the rat, and in man, five distinct amino acid sequence elements (or "domains") have been identified which may be found expressed as part of the 90 kD core CD44 protein (G unthert, U. et al., Cell 65:13-24 (1991); Hoffman, M. et al., Cancer Res. 51:5292-5297 (1991); Jackson, D. G. et al., J. Biol. Chem. 26:4732-4739 (1992); Kugelman, L. et al., J. Invest. Dermatol. 99:381-385 (1992)). These domains are encoded by at least ten distinct exons named v1-v10 (Arch, R. et al., Science 257:682-685 (1992)). CD44 molecules containing one or more of these exons within the common protein backbone are designated as variant forms to distinguish them from the major 90 kD lymphocyte form (standard). Herein, the term exon v6 will be used for nucleotides 1140-1267 of the largest known form of human CD44 (Hoffman, M. et al., Cancer Res. 51:5292-5297 (1991)).
CD44 isoforms play important and distinct roles in tumor invasiveness and metastasis. The standard 90 kD lymphocyte form apparently contributes to the metastatic capacity of non-Hodgkin lymphoma cells in man (Horst, E. et al., Leukemia 4:595-599 (1990); Jalkanen, S. et al., J. Can. Invest. 87:1835-1840 (1991)). The lymphocyte form, but not the 150 kD epithelial form (containing exons 8-10 according to the exon nomenclature), also enhances local tumor formation and the metastatic potential of transfected lymphoma cells in a nude mouse model (Sy, M. S. et al., J. Exp. Med. 174:859-866 (1991)). Expression of the epithelial form is increased in carcinoma cell lines, which may suggest a role in tissue invasiveness (Stamenkovic, I. et al., Cell 56:1057-1062 (1989)). Finally, using a monoclonal antibody which recognizes rat variant CD44, Herrlich reported a direct correlation between CD44 expression and the metastasis of adenocarcinoma cells (PCT International Appl. No. WO 91/17248; see also, G unthert, U. et al., Cell 65:13-24 (1991)).
In man, analysis of the expression pattern of the variant forms of CD44 has been limited to studies on the CD44 variant mRNAs present in cells (Hoffman, M. et al., Cancer Res. 51:5292-5297 (1991)). This is because, prior to the present invention, there were no monoclonal antibodies available capable of distinguishing between the standard and variant CD44s in man. Studies which rely upon mRNA levels as an indicator of protein expression are inherently unreliable due to the fact that mRNAs may or may not be translated. As a result, prior to the present invention, it was not known what kind of tissue distribution the variant forms of CD44 exhibited in man. Moreover, it was not clear, how expression of different isoforms was regulated during normal cell differentiation or what kind of changes in expression might accompany pathological conditions such as malignant transformation, metastasis or inflammatory diseases.
The inventors have produced monoclonal antibodies against exon v6 of the human variant CD44. Distribution of exon v6 containing forms of CD44 (CD44v6 ) in normal tissues and tumors was determined, and compared to that of the forms recognized by mAb Hermes-3, an antibody recognizing the 90 kD CD44 core protein. The results indicate that antibodies which specifically to recognize the amino acid sequence encoded by exon v6 can be used in the detection of malignant transformation and in assessing the metastatic potential of transformed cells. Other results indicate that such antibodies can be used to detect the presence of inflammatory diseases in people.